Disadvantages of randomly induced mutations for reverse genetics • Mutations induced by insertion, chemical, and physical mutagens are randomly distributed along the genome • Large number of mutants (more than number of genes) are needed to be created so that all genes are covered • Difficulty or time/labor consuming in identifying the mutation sites as well as in isolating mutants for every gene Site specific mutation • Target mutagenesis is a method that is used to make specific and intentional changes to the DNA sequence of a target gene • Posttranscriptional gene silencing - Antisense RNA - RNA interference (RNAi) • Homologous recombination (HR) • Gene or genome editing - zinc-finger nucleases (ZFNs) - Transcription activator-like effector nucleases (TALENs) - CRISPR-Cas9 Antisense RNA • Antisense RNA (asRNA), also referred to as antisense transcript, is transcribed from the lagging strand of a gene and is complementary to a specific mRNA or sense transcript with which it hybridizes, and thereby block its translation into protein • asRNA occur naturally and the primary function is to regulate their own gene expression • Notably, more than 30% of annotated transcripts in humans have antisense transcription Pelechano and Steinmetz, 2013 Antisense RNA • Antisense transcripts can be classified into short (<200 nucleotides) and long (>200 nucleotides) non-coding RNAs (ncRNAs) • Antisense RNAs can be produced synthetically and have found wide spread use as research tools for gene silencing • https://en.wikipedia.org/wiki/ Antisense_RNA Using antisense RNA to study gene function --inhibition of carotenoid biosynthesis in transgenic tomatoes • Find a candidate gene • During tomato fruit ripening the expression levels of many genes, encoding specific enzyme responsible for the physical changes in the fruit increase • Several gene cDNA that correspond to ripenning-enhanced mRNAs identified, however function of the encoded protein is unknown • pTOM5 may be involved in carotenoid biosynthesis • In this study, antisense RNA to show that the pTOM5 gene product is part of this biosynthetic pathway Inhibition of carotenoid biosynthesis in transgenic tomatoes • A chimeric pTOM5 antisens gene was constructed and then transferred into tomato plants an antisense gene mutation by using Agrobacterium • Transformed plants have corolla with less intense yellow pigmentation Inhibition of carotenoid biosynthesis in transgenic tomatoes • The yellow fruit have drastically reduced levels of carotenoids, particular lycopene, compared to normal ripening fruit, indicating that the protein encoded by pTOM5 mRNA is involved in either the synthesis or regulation of synthesis of carotenoids in the fruit Development and commercialization of reduced lignin • Alfalfa (Medicago sativa) is the most cultivated forage legume in the world • Genetically engineered (GE) glyphosate-resistant alfalfa is deregulated in 2010 • Since 2015, an alfalfa variety with reduced lignin (RL) has been available as a stacked trait with glyphosate resistance • Approximately 15% of the alfalfa currently grown in the U.S. is GE, and this is expected to grow to up to 50% within the next 10 years Barros et al., 2018 Development and commercialization of reduced lignin • Lignin reduction was achieved by downregulating the gene encoding caffeoyl-CoA 3-O- methyltransferase (CCoAOMT), and development of the commercial product, branded as HarvXtra Barros et al., 2018 Development and commercialization of reduced lignin a) With a 5–10 days harvest delay, RL alfalfa harvested on a 35–40 days harvest interval showed 20% gain in forage mass and no significant reduction in RFQ compared to reference cultivars harvested on a 30-day harvest interval b) Reducing the cutting frequency improved the persistence of the stand Barros et al., 2018 microRNA • A short (20-30 nt) noncoding RNA • Negatively regulate gene expression by pairing to mRNAs of protein-coding genes to direct their post-transcriptional repression First identified microRNA --lin-4 in C. elegans • lin-4 and lin-14, induced mutants, show abnormal larva development from L1 to L2 • lin-14 is a protein coding gene Lin-4 Lin-14 (12h) (7h) (8h) (14h) (10h) Lee et al., 1993 First identified microRNA --lin-4 in C. elegans • lin-4 does not encode a protein, but encodes a pair of small RNA • A 21 nt of lin-4 RNA complementary to a repeated sequence element in the 3︎’ UTR of lin-14 mRNA • lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction, reduce lin-14 protein level without change in level of lin-14 mRNA Lee et al., 1993 First identified microRNA --lin-4 in C. elegans • Let-7, another gene in the C. elegans, encodes a 22 bp regulatory RNA found in 2000, which promotes the transition from L4 to adult • Hundreds of microRNA genes and families were identified in diverse organisms lin-4 lin-14 (12h) (7h) (8h) (14h) (10h) let-7 lin-41 Lee et al., 1993; Reinhart et al., 2000 microRNA regulates gene expression • MicroRNAs (miRNAs) assemble with Argonaute proteins into miRNA-induced silencing complexes (miRISCs) to direct post- transcriptional silencing of complementary mRNA targets • miRNAs silence gene expression by repressing translation and accelerating target mRNA degradation • microRNA regulates gene expression microRNA recognize sites • Conserved pairing to the seed region (7-8 nt) of microRNA Bartel, 2004, 2009 microRNA identified in diverse species • In human, more than 5,300 genes involved in diverse biological functions/processes are targeted/regulated by those small RNAs – Cell fate (tissue development) – Cancer • In plants, genes regulated by microRNA are involved in development in plants and abiotic stress Bartel, 2009 Example 1: MicroRNA control of PHABULOSA in leaf development Ile Gly Met Lys Pro Gly Pro Asp ------AUU GGG AUG AAG CCU GGU CCG GAU------ CCC UAC UUC GGA CCA GGC miR166 Gly (GGU) Asp (GAU) Gly (GGA) Mallory et al., 2004 Example 2: MicroRNA control of PHABULOSA in leaf development • Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA- guided cleavage of these mRNAs in vitro (performed or taking place in a test tube, culture dish, or elsewhere outside a living organism). • Confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. Artificial microRNA (amiRNA) for a specific gene silencing to study gene function • http://wmd3.weigelworld.org/cgi-bin/webapp.cgi • This tool was initially implemented for Arabidopsis thaliana, but has now been extended to >90 additional species Schwab et al., The Plant Cell 2006 Example 1: Gene silencing of FT using amiRNA • amiR-ft-1 targets FT, a gene related to promotion of flowering • The fragment was placed behind a promoter • Transgenic plants were generated by Agrobacterium mediated transformation Wild type -10 amiR-3-1 Schwab et al., The Plant Cell 2006 MicroRNA and gene therapy • First FDA approved RNAi therapy Hereditary Transthyretin (TTR) amyloidosis (ATTR amyloidosis) • Hereditary transthyretin amyloidosis is an autosomal dominant, multisystemic, progressive, life-threatening disease caused from deposition of insoluble ATTR amyloid fibrils in various organs and tissues • Hereditary transthyretin amyloidosis is inexorably progressive, with survival of 2 to 15 years after the onset of neuropathy • TTR is predominantly produced by hepatocytes and circulates as a homotetrameric complex that functions as a transporter for thyroxine and vitamin A • However, TTR monomers can undergo a major conformational transformation to aggregate in a highly ordered and abnormal amyloid fibril form (ATTR); Deposition of wild-type (wt) ATTR typically occurs in older patients, giving rise to wt-ATTR amyloidosis, formerly known as senile systemic amyloidosis Gofieau et al., 1996; Giaever et al., 2014 Hereditary Transthyretin (TTR) amyloidosis (ATTR amyloidosis) • Mutations in the TTR gene give rise to variants that destabilize the tetramer such that TTR can more readily undergo the conformational change to amyloid, and these genetic changes underlie the various hereditary ATTR amyloidosis (h- ATTR amyloidosis) clinical syndromes. The liver is the primary source of circulating tetrameric transthyretin protein • The liver is the primary source of circulating tetrameric transthyretin protein • Current treatment options for hereditary transthyretin amyloidosis are limited and include orthotopic liver transplantation and transthyretin tetramer stabilizers (tafamidis or diflunisal). However, many patients who are treated with these approaches continue to have disease progression • Patisiran, a hepatically directed investigational RNAi therapeutic agent, harnesses this process to reduce the production of mutant and wild-type transthyretin by targeting the 3′ untranslated region of transthyretin mRNA Homologous Recombination (HR) induce targeted gene mutation Example 1: Yeast deletion project • Life with 6000 genes – Yeast, single cell, first genome sequence of an eukaryote – Genome size of 12 Mb and 5,885 protein-coding genes • Saccharomyces Genome Deletion Project using homologous recombination (HR) – A complete, systematic deletion collection to identify essential genes and understand gene function • http://www-sequence.stanford.edu/group/ yeast_deletion_project/deletions3.html Gofieau et al., 1996; Giaever et al., 2014 Example 1: Yeast deletion project • More than 21,000 mutant strains that carry precise deletions of 6,000 open reading frames • The yeast deletion